Method and pharmaceutical composition for use in the treatment of cancer

ABSTRACT

The present invention relates to a soluble peptide comprising the amino acids sequence: KRFYVVMWKK (SEQ ID NO: 1) or a function-conservative variant thereof for use in the treatment of cancer. The invention also relates to a pharmaceutical composition for use in the treatment of cancer comprising at least one soluble peptide according to the invention or at least one acid nucleic according to the invention or at least one expression vector according to the invention, or at least one host cell according to the invention and a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

The present invention relates to a soluble peptide comprising the aminoacids sequence: KRFYVVMWKK (SEQ ID NO:1) or a function-conservativevariant thereof for use in the treatment of cancer.

The invention also relates to a pharmaceutical composition for use inthe treatment of cancer comprising at least one soluble peptideaccording to the invention or at least one acid nucleic according to theinvention or at least one expression vector according to the invention,or at least one host cell according to the invention and apharmaceutically acceptable carrier.

BACKGROUND OF THE INVENTION

Cancer is a malignant neoplasm, is a broad group of various diseases,all involving unregulated cell growth. In 2007, cancer caused about 13%of all human deaths worldwide (7.9 million). Rates are rising as morepeople live to an old age and as mass lifestyle changes occur in thedeveloping world.

Particularly, Chronic Lymphocytic Leukemia (CLL) is the most commonadult leukemia in the Western countries and is characterized by aprogressive accumulation of monoclonal CD5⁺ B-lymphocytes in theperipheral blood, bone marrow, and secondary lymphoid organs. Theresulting congestion leads to the progressive failure of the immune andhematopoietic systems. High-risk hallmarks predictive of CLL progressioninclude the cytogenetic features' mutation/deletion of 17p13 (TP53) and11q22-q23 (ATM), IGHV unmutated status, high expression of ZAP70, CD38,soluble CD23 increase and the currently studied and not still validatedmutations in NOTCH1, MYD88, BIRC3, XPO1, KLHL6, SF3B1, and POT1 genes[Gribben J G, 2010; Lanasa M C, 2010 and Chiorazzi N. et al., 2005].Patients with dysfunction relevant to ATM and TP53 genes have thepoorest prognosis requiring specific aggressive therapy includingallogenic stem cell transplantation [Pospisilova N, 2012]. Thecharacteristics of CLL are: (i) Incurable, as all patients willeventually relapse, underscoring a resistance of the disease to currenttreatment options. (ii) Very heterogeneous disease in terms of responseto the—yet non-optimal—existing treatments. (iii) Drug resistanceremains a major cause of treatment failure in CLL and its inevitablefate due to the prolonged natural course of the disease and the repeatedtreatments, creating a relevant social and health problem. (iv) Mainlyaffects elderly people and is considered a paradigmatic example of mostage-related cancers. (v) Robust and specific markers predictive ofresponse to treatment are still lacking, though urgently needed in orderto implement risk-adapted, personalized treatment and maximize clinicalbenefit while minimizing costs.

Even though the direct cause for the development of this malignancy isnot fully understood, it is now well demonstrated that CLL represents aperfect example of a human malignancy caused by an imbalance betweenproliferation and Programmed Cell Death (PCD) [Chiorazzi N, 2007]. Thus,a better understanding of PCD mechanisms regulating the lifespan of theleukemic CLL cells should provide key advances for therapeuticinterventions in this leukemia.

PCD is a self-destruction process characterized by stereotypedultrastructural changes including mitochondrial alterations,condensation of the nucleus and cytoplasm, membrane blebbing andexternal display of phosphatidylserine. Intense research performed inthe last decade has identified a multitude of enzymes and otherregulatory proteins involved in the modulation of PCD. These studiesconclude that, in most cases, PCD occurs when a family of cysteineproteases, known as caspases, is activated. Since the induction ofapoptosis through the use of caspase activators may theoreticallyconstitute a treatment for cancer, the initial pro-apoptotic anti-cancertrials have focused on caspase activity. Unfortunately, most of thesestudies are still in preclinical development because of their lowefficacy. In part, this may be due to the fact that PCD can proceed evenwhen the caspase cascade is blocked. This fact has revealed theexistence of an alternative pathway defined as caspase-independent. Acomprehensive analysis of caspase-independent PCD pathways offerstherefore a new challenge in the design of therapeutic strategiesagainst CLL and other neoplastic diseases.

As indicated above, drug resistance remains a major cause of treatmentfailure in CLL. In fact, current therapies are responsible for severalside effects, increasing the occurrence of treatment-relateddisabilities that may ultimately affect the well-being, if not thesurvival rate, of most patients. Until now, the goal of therapy has beento maintain the best quality of life and start treatment only whenpatients became symptomatic from their disease. For the majority ofpatients this means following a “wait-and-see” approach to determine therate of progression of the disease and assess the development ofsymptoms. Initial treatments for CLL patients have included either anucleosid analog (Fludarabine) or an alkylating agent (Chlorambucil).This initial approach has been improved by combination regimens such asfludarabine and cyclophosphamide (FC), or more recently by the additionof rituximab to FC (FCR treatment) that is now accepted as the standardfront-line therapy. Alternative treatments have been developed forresistant patients or in relapse such as bendamustine, proteasomeinhibitors, or monoclonal antibodies (anti-CD52, optimized anti-CD20,anti-CD23, etc.).

Concerning the current clinical trials, the more relevant are the use ofmonoclonal antibodies (GA101, lumiliximab, lucatumumab), BH3 mimetics(obatoclax, ABT-263), cyclin-dependent kinase inhibitors (flavopiridol,SNS-032), Lyn-kinase inhibitors (dasatinib, bafetinib), hypomethylatingagents (azacytidine, decitabine), histone deacetylase inhibitors(parobinastat), purine analogs (8-chloroadenosine, forodesine), andsmall modular immunopharmaceuticals (TRU-016). Molecules inhibitingdownstream signaling after B-cell receptor ligation are novel oralagents interacting at different targets including phosphatidylinositol3-kinase inhibitors (CAL-101), Bruton's tyrosine kinase (BTK)(PCI-32765), and Spleen Tyrosine Kinase (SYK)-inhibitors (fostamitinib).

Most of the above-described chemotherapeutic treatments inducecytotoxicity via a caspase-dependent mechanism (see above, page 2) witha quite variable outcome, with many patients having a positive reactionwhereas others remain refractory (15-25% of CLL patients becomerefractory during the course of the disease). Indeed, as leukemic Bcells present molecular defects that make them particularly resistant tothe caspase-dependent PCD pathway (p53 inactivation, overexpression ofanti-apoptotic proteins, such as Mcl-1 or Bcl-2), a significant group ofCLL patients are refractory to the current chemotherapeutic treatments.For that reason, the introduction of new drugs that induce PCD viaalternative caspase-independent PCD pathways could provide new means ofimproving the current therapeutic strategies used in CLL treatment.

The CD47 receptor is a widely expressed member of the immunoglobulin(Ig) superfamily, functioning both as a receptor for thrombospondin-1(TSP-1) and as a ligand for the transmembrane signal regulatory proteinsSIRP α and γ [Brown E J et al., 2001]. These molecules regulate variousbiological phenomena in the immune system, including plateletactivation, leukocyte migration, macrophage multinucleation, and PCD.Neither SIRP α nor SIRP γ has been implicated in CD47-induced PCD incontrast to TSP-1, which has been shown to bind CD47 specifically viaits COOH-terminal cell-binding domain. Many cancers appear to upregulateCD47 as a mechanism of immune evasion and recent work showed that CD47is a prognostic factor and a potential therapeutic target in differenttypes of Non-Hodgkin Lymphomas (NHL), including CLL [Edris, B et al.,2012; Willingham, S. B et al., 2012; Chao, M. P et al., 2010; Jaiswal, Set al., 2009 and Chao, M. P et al., 2011]. The inventors and others haverecently demonstrated that CD47 ligation, by an immobilized anti-CD47mAb (not by a soluble anti-CD47), induces caspase-independent PCD, evenin CLL cells from refractory patients [Mateo V et al., 1999; Roue G etal., 2003; Barbier S et al., 2009; Merle-Beral H et al., 2009; Bras M etal., 2007; Mateo V et al., 2002].

SUMMARY OF THE INVENTION

The inventors demonstrate that CD47 ligation by 4N1K (SEQ ID NO:1), asoluble and monovalent decapeptide that mimics the C-terminal domain ofTSP-1, induces caspase-independent PCD in B-chronic lymphocytic leukemia(CLL) primary cells. Contrary to the anti-CD47 mAb which needs to beimmobilized to induced PCD, the soluble 4N1K peptide does not need to becoated on plastic to induce caspase-independent PCD. A negative controlpeptide 4NGG (SEQ ID NO:2-4N1K mutated in two amino acids-) is unable toinduce PCD, signifying the specificity of the 4N1K PCD induction (FIG.1). Moreover, CD47 ligation by 4N1K and its derivative PKHB1specifically eliminates leukemic B-cells, and not healthy B-lymphocytesor resting normal B-cells from CLL patients (FIGS. 2, 5 and 6) andrepresents a better means of inducing death than caspase-dependent PCD(this form of death is effective even in CLL cells from drug refractoryindividuals bearing deletion on 17p13 or 11q22-q23: ATM/TP53inactivated-, FIG. 2). In vivo mouse studies fully confirm thespecificity of this peptide strategy in inducing PCD in leukemic cells(FIGS. 7 and 8).

Thus, the invention relates to a soluble peptide comprising the aminoacids sequence: KRFYVVMWKK (SEQ ID NO:1) or a function-conservativevariant thereof for use in the treatment of cancer.

The invention also relates to a pharmaceutical composition for use inthe treatment of cancer comprising at least one soluble peptideaccording to the invention or at least one acid nucleic according to theinvention or at least one expression vector according to the invention,or at least one host cell according to the invention and apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION Peptide and Uses Thereof

A first object of the invention relates to a soluble peptide comprisingthe amino acids sequence: KRFYVVMWKK (SEQ ID NO:1) or afunction-conservative variant thereof for use in the treatment ofcancer.

The invention also encompasses peptides that are function-conservativevariants of the soluble peptide comprising SEQ ID NO: 1 as describedhere above.

In one embodiment, the soluble peptide according to the invention maydiffer from 1, 2 or 3 amino acids to the SEQ ID NO:1.

In another embodiment, the soluble peptide according to the inventionmay differ from 4 or 5 amino acids to the SEQ ID NO:1.

In one embodiment, the soluble peptide of the invention comprises atleast 75% identity over said the SEQ ID NO: 1, even more preferably atleast 80%, at least 85%, at least 90%, at least 95%, at least 97% and isstill able to decrease tumor cell proliferation or still able to inducePCD in tumor cell.

In one embodiment, the soluble peptide of the invention consists in theamino acid sequence as set forth in SEQ ID NO:1 or a variant thereofcomprising at least 75%, preferably at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.5% or 99.9% identity with SEQ ID NO:1 and is stillable to decrease tumor cell proliferation or still able to induce PCD intumor cells.

To verify whether the newly generated soluble peptides induce the sametype of caspase-independent PCD than the initially characterized peptide4N1K a flow cytometry analysis (such as described in FIG. 2) may beperformed with each peptide. A comparison of the results obtained intreatments with/without the caspase inhibitor Z-VAD.fmk will corroboratethat the mode of cell death induced by the 4N1K-derived peptides iscaspase-independent. Additionally, a time-course and a dose-responseperformed in different tumor cells will determine the optimal conditionsfor each peptide and each malignant cell type.

In one embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 50 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

In another embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 45 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

In another embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 40 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

In another embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 30 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

In another embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 20 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

In another embodiment of the invention, said soluble peptide is an aminoacid sequence of less than 15 amino acids long that comprises the aminoacid sequence SEQ ID NO:1 as defined here above.

As used herein, the term “Function-conservative variants” refer to thosein which a given amino acid residue in a protein or enzyme has beenchanged (inserted, deleted or substituted) without altering the overallconformation and function of the peptide. Such variants include proteinhaving amino acid alterations such as deletions, insertions and/orsubstitutions. A “deletion” refers to the absence of one or more aminoacids in the protein. An “insertion” refers to the addition of one ormore of amino acids in the protein. A “substitution” refers to thereplacement of one or more amino acids by another amino acid residue inthe protein. Typically, a given amino acid is replaced by an amino acidhaving similar properties (such as, for example, polarity, hydrogenbonding potential, acidic, basic, hydrophobic, aromatic, and the like).This given amino acid can be a natural amino acid or a non natural aminoacid. Amino acids other than those indicated as conserved may differ ina protein so that the percent protein or amino acid sequence similaritybetween any two proteins of similar function may vary and may be, forexample, from 70% to 99% as determined according to an alignment schemesuch as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. A “function-conservative variant” also includes apolypeptide which has at least 60% amino acid identity as determined byBLAST or FASTA algorithms, preferably at least 75%, more preferably atleast 85%, still preferably at least 90%, and even more preferably atleast 95%, and which has the same or substantially similar properties orfunctions as the native or parent protein to which it is compared. Twoamino acid sequences are “substantially homologous” or “substantiallysimilar” when greater than 80%, preferably greater than 85%, preferablygreater than 90% of the amino acids are identical, or greater than about90%, preferably greater than 95%, are similar (functionally identical)over the whole length of the shorter sequence. Preferably, the similaror homologous sequences are identified by alignment using, for example,the GCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wis.) pileup program, or any of sequence comparisonalgorithms such as BLAST, FASTA, etc.

Typically, the invention encompasses soluble peptides substantiallyidentical to the soluble peptide comprising SEQ ID NO:1 in which one ormore residues have been conservatively substituted with a functionallysimilar residue and which displays the functional aspects of the solublepeptides comprising SEQ ID NO:1 as described here above, i.e. beingstill able to decrease tumor cell proliferation in substantially thesame way as a peptide consisting of the given amino acid sequence.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid or another.

The term “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject peptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Chemical derivatives also include peptides that contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For examples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine. The term “conservativesubstitution” also includes the use of non natural amino acids aimed tocontrol and stabilize peptides or proteins secondary structures. Thesenon natural amino acids are chemically modified amino acids such asprolinoamino acids, beta-amino acids, N-methylamino acids,cyclopropylamino acids, alpha,alpha-substituted amino acids as describehere below. These non natural amino acids may include also fluorinated,chlorinated, brominated- or iodinated modified amino acids.

In one embodiment, soluble peptides of the invention may be as describedin example 2.

In another embodiment, the soluble peptide of the invention is the PKHB1peptide as describe in examples 2 and 3.

In another embodiment, the soluble peptide of the invention is the PKHB3peptide as describe in example 2.

In another embodiment, the soluble peptide of the invention is the PKHB4peptide as describe in example 2.

In another embodiment, the soluble peptide of the invention is the PKHB9peptide as describe in example 2.

In another embodiment, the soluble peptide of the invention is thePKHB10 peptide as describe in example 2.

In another embodiment, the soluble peptide of the invention is thePKHB11 peptide as describe in example 2.

In one embodiment, soluble peptides of the invention may comprise a tag.A tag is an epitope-containing sequence which can be useful for thepurification of the soluble peptides. It is attached to by a variety oftechniques such as affinity chromatography, for the localization of saidpeptide or polypeptide within a cell or a tissue sample usingimmunolabeling techniques, the detection of said peptide or polypeptideby immunoblotting etc. Examples of tags commonly employed in the art arethe GST (glutathion-S-transferase)-tag, the FLAG™-tag, the Strep-Tag™,V5 tag, myc tag, His tag etc.

In one embodiment, soluble peptides of the invention may be labelled bya fluorescent dye. Dye-labelled fluorescent peptides are important toolsin cellular studies. Peptides can be labelled on the N-terminal side oron the C-terminal side.

N-Terminal Peptide Labeling Using Amine-Reactive Fluorescent Dyes:

Amine-reactive fluorescent probes are widely used to modify peptides atthe N-terminal or lysine residue. A number of fluorescent amino-reactivedyes have been developed to label various peptides, and the resultantconjugates are widely used in biological applications. Three majorclasses of amine-reactive fluorescent reagents are currently used tolabel peptides: succinimidyl esters (SE), isothiocyanates and sulfonylchlorides.

C-Terminal Labeling Using Amine-Containing Fluorescent Dyes:

Amine-containing dyes are used to modify peptides using water-solublecarbodiimides (such as EDC) to convert the carboxy groups of thepeptides into amide groups. Either NHS or NHSS may be used to improvethe coupling efficiency of EDC-mediated protein-carboxylic acidconjugations.

Labelled peptides derived from 4N1K have the following general formula:

Where X and/or Y can be nothing or hydrogen and/or spacers and/orfluorescent dyes.

PKHB8 (formula (VII)) is an example of peptide from this series where aspacer formed by two beta-alanine residues and a fluorescent dye(fluorescein) have been introduced on the N-terminal side of thepeptide, on the alpha-amino group of the lysine residue:

In another embodiment, the soluble peptide of the invention is the PKHB8peptide.

In specific embodiments, it is contemplated that soluble peptides usedin the therapeutic methods of the present invention may be modified inorder to improve their therapeutic efficacy. Such modification oftherapeutic compounds may be used to decrease toxicity, increasecirculatory time, or modify biodistribution. For example, the toxicityof potentially important therapeutic compounds can be decreasedsignificantly by combination with a variety of drug carrier vehiclesthat modify biodistribution.

A strategy for improving drug viability is the utilization ofwater-soluble polymers. Various water-soluble polymers have been shownto modify biodistribution, improve the mode of cellular uptake, changethe permeability through physiological barriers; and modify the rate ofclearance from the body. To achieve either a targeting orsustained-release effect, water-soluble polymers have been synthesizedthat contain drug moieties as terminal groups, as part of the backbone,or as pendent groups on the polymer chain.

Polyethylene glycol (PEG) has been widely used as a drug carrier, givenits high degree of biocompatibility and ease of modification. Attachmentto various drugs, proteins, and liposomes has been shown to improveresidence time and decrease toxicity. PEG can be coupled to activeagents through the hydroxyl groups at the ends of the chain and viaother chemical methods; however, PEG itself is limited to at most twoactive agents per molecule. In a different approach, copolymers of PEGand amino acids were explored as novel biomaterials which would retainthe biocompatibility properties of PEG, but which would have the addedadvantage of numerous attachment points per molecule (providing greaterdrug loading), and which could be synthetically designed to suit avariety of applications.

Those of skill in the art are aware of PEGylation techniques for theeffective modification of drugs. For example, drug delivery polymersthat consist of alternating polymers of PEG and tri-functional monomerssuch as lysine have been used by VectraMed (Plainsboro, N.J.). The PEGchains (typically 2000 daltons or less) are linked to the a- and e-aminogroups of lysine through stable urethane linkages. Such copolymersretain the desirable properties of PEG, while providing reactive pendentgroups (the carboxylic acid groups of lysine) at strictly controlled andpredetermined intervals along the polymer chain. The reactive pendentgroups can be used for derivatization, cross-linking, or conjugationwith other molecules. These polymers are useful in producing stable,long-circulating pro-drugs by varying the molecular weight of thepolymer, the molecular weight of the PEG segments, and the cleavablelinkage between the drug and the polymer. The molecular weight of thePEG segments affects the spacing of the drug/linking group complex andthe amount of drug per molecular weight of conjugate (smaller PEGsegments provides greater drug loading). In general, increasing theoverall molecular weight of the block co-polymer conjugate will increasethe circulatory half-life of the conjugate. Nevertheless, the conjugatemust either be readily degradable or have a molecular weight below thethreshold-limiting glomular filtration (e.g., less than 45 kDa).

In addition, to the polymer backbone being important in maintainingcirculatory half-life, and biodistribution, linkers may be used tomaintain the therapeutic agent in a pro-drug form until released fromthe backbone polymer by a specific trigger, typically enzyme activity inthe targeted tissue. For example, this type of tissue activated drugdelivery is particularly useful where delivery to a specific site ofbiodistribution is required and the therapeutic agent is released at ornear the site of pathology. Linking group libraries for use in activateddrug delivery are known to those of skill in the art and may be based onenzyme kinetics, prevalence of active enzyme, and cleavage specificityof the selected disease-specific enzymes (see e.g., technologies ofestablished by VectraMed, Plainsboro, N.J.). Such linkers may be used inmodifying the soluble peptides-derived described herein for therapeuticdelivery.

According to the invention, soluble peptides may be produced byconventional automated peptide synthesis methods or by recombinantexpression. General principles for designing and making proteins arewell known to those of skill in the art.

Soluble peptides of the invention may be synthesized in solution or on asolid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols as described in Stewart and Young; Tamet al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross andMeienhofer, 1979. Soluble peptides of the invention may also besynthesized by solid-phase technology employing an exemplary peptidesynthesizer such as a Model 433A from Applied Biosystems Inc. The purityof any given protein; generated through automated peptide synthesis orthrough recombinant methods may be determined using reverse phase HPLCanalysis. Chemical authenticity of each peptide may be established byany method well known to those of skill in the art.

As an alternative to automated peptide synthesis, recombinant DNAtechnology may be employed wherein a nucleotide sequence which encodes aprotein of choice is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression as described herein below.Recombinant methods are especially preferred for producing longerpolypeptides.

A variety of expression vector/host systems may be utilized to containand express the peptide or protein coding sequence. These include butare not limited to microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid or cosmid DNA expression vectors;yeast transformed with yeast expression vectors (Giga-Hama et al.,1999); insect cell systems infected with virus expression vectors (e.g.,baculovirus, see Ghosh et al., 2002); plant cell systems transfectedwith virus expression vectors (e.g., cauliflower mosaic virus, CaMV;tobacco mosaic virus, TMV) or transformed with bacterial expressionvectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); oranimal cell systems. Those of skill in the art are aware of varioustechniques for optimizing mammalian expression of proteins, see e.g.,Kaufman, 2000; Colosimo et al., 2000. Mammalian cells that are useful inrecombinant protein productions include but are not limited to VEROcells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells(such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and293 cells. Exemplary protocols for the recombinant expression of thepeptide substrates or fusion polypeptides in bacteria, yeast and otherinvertebrates are known to those of skill in the art and a brieflydescribed herein below. U.S. Pat. No. 6,569,645; U.S. Pat. No.6,043,344; U.S. Pat. No. 6,074,849; and U.S. Pat. No. 6,579,520 providespecific examples for the recombinant production of soluble peptides andthese patents are expressly incorporated herein by reference for thoseteachings. Mammalian host systems for the expression of recombinantproteins also are well known to those of skill in the art. Host cellstrains may be chosen for a particular ability to process the expressedprotein or produce certain post-translation modifications that will beuseful in providing protein activity. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be important for correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, 293, WI38, andthe like have specific cellular machinery and characteristic mechanismsfor such post-translational activities and may be chosen to ensure thecorrect modification and processing of the introduced, foreign protein.

In the recombinant production of the soluble peptides-derived of theinvention, it would be necessary to employ vectors comprisingpolynucleotide molecules for encoding the soluble peptides-derived.Methods of preparing such vectors as well as producing host cellstransformed with such vectors are well known to those skilled in theart. The polynucleotide molecules used in such an endeavor may be joinedto a vector, which generally includes a selectable marker and an originof replication, for propagation in a host. These elements of theexpression constructs are well known to those of skill in the art.Generally, the expression vectors include DNA encoding the given proteinbeing operably linked to suitable transcriptional or translationalregulatory sequences, such as those derived from a mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, mRNA ribosomalbinding sites, and appropriate sequences which control transcription andtranslation.

The terms “expression vector,” “expression construct” or “expressioncassette” are used interchangeably throughout this specification and aremeant to include any type of genetic construct containing a nucleic acidcoding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed.

The choice of a suitable expression vector for expression of thepeptides or polypeptides of the invention will of course depend upon thespecific host cell to be used, and is within the skill of the ordinaryartisan. Methods for the construction of mammalian expression vectorsare disclosed, for example, in Okayama and Berg, 1983; Cosman et al.,1986; Cosman et al., 1984; EP-A-0367566; and WO 91/18982. Otherconsiderations for producing expression vectors are detailed in e.g.,Makrides et al., 1999; Kost et al., 1999. Wurm et al., 1999 isincorporated herein as teaching factors for consideration in thelarge-scale transient expression in mammalian cells for recombinantprotein production.

Expression requires that appropriate signals be provided in the vectors,such as enhancers/promoters from both viral and mammalian sources thatmay be used to drive expression of the nucleic acids of interest in hostcells. Usually, the nucleic acid being expressed is undertranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. Nucleotide sequences are operably linked whenthe regulatory sequence functionally relates to the DNA encoding thepeptide of interest (i.e., 4N1K, a variant and the like). Thus, apromoter nucleotide sequence is operably linked to a given DNA sequenceif the promoter nucleotide sequence directs the transcription of thesequence.

Similarly, the phrase “under transcriptional control” means that thepromoter is in the correct location and orientation in relation to thenucleic acid to control RNA polymerase initiation and expression of thegene. Any promoter that will drive the expression of the nucleic acidmay be used. The particular promoter employed to control the expressionof a nucleic acid sequence of interest is not believed to be important,so long as it is capable of directing the expression of the nucleic acidin the targeted cell. Thus, where a human cell is targeted, it ispreferable to position the nucleic acid coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter. Common promoters include, e.g., the humancytomegalovirus (CMV) immediate early gene promoter, the SV40 earlypromoter, the Rous sarcoma virus long terminal repeat, [beta]-actin, ratinsulin promoter, the phosphoglycerol kinase promoter andglyceraldehyde-3-phosphate dehydrogenase promoter, all of which arepromoters well known and readily available to those of skill in the art,can be used to obtain high-level expression of the coding sequence ofinterest. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa coding sequence of interest is contemplated as well, provided that thelevels of expression are sufficient to produce a recoverable yield ofprotein of interest. By employing a promoter with well known properties,the level and pattern of expression of the protein of interest followingtransfection or transformation can be optimized. Inducible promotersalso may be used.

Another regulatory element that is used in protein expression is anenhancer. These are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Where an expression construct employs a cDNA insert, one will typicallydesire to include a polyadenylation signal sequence to effect properpolyadenylation of the gene transcript. Any polyadenylation signalsequence recognized by cells of the selected transgenic animal speciesis suitable for the practice of the invention, such as human or bovinegrowth hormone and SV40 polyadenylation signals.

Acids Nucleic, Vectors, Recombinant Host Cells and Uses Thereof

Another object of the invention relates to a nucleic acid encoding anamino acids sequence comprising SEQ ID NO: 1 or a function-conservativevariant thereof as described here above for use in the prevention ortreatment of cancer.

In one embodiment, said nucleic acid encoding an amino acids sequenceconsisting on SEQ ID NO: 1.

Nucleic acids of the invention may be produced by any technique knownper se in the art, such as, without limitation, any chemical,biological, genetic or enzymatic technique, either alone or incombination(s).

Another object of the invention is an expression vector comprising anucleic acid sequence encoding an amino sequence comprising SEQ ID NO: 1or a function-conservative variant thereof as described here above foruse in the prevention or treatment of cancer.

According to the invention, expression vectors suitable for use in theinvention may comprise at least one expression control elementoperationally linked to the nucleic acid sequence. The expressioncontrol elements are inserted in the vector to control and regulate theexpression of the nucleic acid sequence. Examples of expression controlelements include, but are not limited to, lac system, operator andpromoter regions of phage lambda, yeast promoters and promoters derivedfrom polyoma, adenovirus, retrovirus, lentivirus or SV40. Additionalpreferred or required operational elements include, but are not limitedto, leader sequence, termination codons, polyadenylation signals and anyother sequences necessary or preferred for the appropriate transcriptionand subsequent translation of the nucleic acid sequence in the hostsystem. It will be understood by one skilled in the art that the correctcombination of required or preferred expression control elements willdepend on the host system chosen. It will further be understood that theexpression vector should contain additional elements necessary for thetransfer and subsequent replication of the expression vector containingthe nucleic acid sequence in the host system. Examples of such elementsinclude, but are not limited to, origins of replication and selectablemarkers. It will further be understood by one skilled in the art thatsuch vectors are easily constructed using conventional methods orcommercially available.

Another object of the invention is a host cell comprising an expressionvector as described here above for use in the prevention or treatment ofcancer.

According to the invention, examples of host cells that may be used areeukaryote cells, such as animal, plant, insect and yeast cells andprokaryotes cells, such as E. coli. The means by which the vectorcarrying the gene may be introduced into the cells include, but are notlimited to, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art.

In another embodiment, eukaryotic expression vectors that function ineukaryotic cells are used. Examples of such vectors include, but are notlimited to, viral vectors such as retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, poxvirus,poliovirus; lentivirus, bacterial expression vectors, plasmids, such aspcDNA3 or the baculovirus transfer vectors. Preferred eukaryotic celllines include, but are not limited to, COS cells, CHO cells, HeLa cells,NIH/3T3 cells, 293 cells (ATCC#CRL1573), T2 cells, dendritic cells, ormonocytes.

Therapeutic Methods

In a particular embodiment, soluble peptides, nucleic acids, expressionvector or host cells of the invention may be useful in the treatment ofa cancer selected form the group consisting of adrenal cortical cancer,anal cancer, bile duct cancer, bladder cancer, bone cancer, brain andcentral nervous system cancer, breast cancer, Castleman disease,cervical cancer, colorectal cancer, endometrial cancer, esophaguscancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin'sdisease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer,laryngeal and hypopharyngeal cancer, liver cancer, lung cancer,mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngealcancer, ovarian cancer, pancreatic cancer, penile cancer, pituitarycancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivarygland cancer, skin cancer, stomach cancer, testicular cancer, thymuscancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterinecancer.

In another particular embodiment, soluble peptides, nucleic acids,expression vector or host cells of the invention may be useful in theprevention and the treatment of leukemia and particularly in acutelymphoblastic leukemia, B-chronic lymphocytic leukemia, hairy-cellleukemia, adult T-cell leukemia, prolymophocytic leukaemia of T-celltype or myeloid leukaemia.

In one embodiment, the leukemia is a B-chronic lymphocytic leukemia(CLL).

In a particular embodiment, soluble peptides, nucleic acids, expressionvector or host cells of the invention may be useful in the treatment ofa refractory CLL. In another particular embodiment, soluble peptides,nucleic acids, expression vector or host cells of the invention may beuseful in the treatment of a refractory CLL with poor prognosis,including unmutated IGHV, complex karyotype and dysfunctional or mutatedTP53, ATM, NOTH1, MYD88, XPO1, KLHL6, SF3B1, POT1 and BIRC3 B-cells.

As used herein, the term “refractory CLL” denotes a CLL refractory tocommon treatments used against leukemia (described in pages 2 and 3).

In a particular embodiment, soluble peptides, nucleic acids, expressionvector or host cells of the invention may be useful in the treatment ofrefractory CLL that present intrinsic mutations that could allow to drugresistance (e.g, mutation/deletions in TP53, ATM, NOTH1, MYD88, XPO1,KLHL6, SF3B1, POT1 and BIRC3 genes or refractory to the treatmentsdescribed in pages 2 and 3).

In a particular embodiment, soluble peptides, nucleic acids, expressionvector or host cells of the invention may be useful in the treatment ofrefractory CLL where common treatment like anti-CD20, fludarabine orcladribine are not working.

Another object of the invention relates to a method for treating cancercomprising administering to a subject in need thereof a therapeuticallyeffective amount of soluble peptides as described above or a nucleicacid according to the invention or an expression vector according to theinvention or a host cell according to the invention.

In one aspect, the invention relates to a method for treating cancercomprising administering to a subject in need thereof a therapeuticallyeffective amount of soluble peptide of SEQ ID NO:1 or afunction-conservative variant thereof as above described.

In another embodiment, the invention relates to a method for treatingcancer comprising administering to a subject in need thereof atherapeutically effective amount of a soluble peptide according to theinvention.

As used herein, the term “therapeutically effective amount” is intendedfor a minimal amount of active agent, which is necessary to imparttherapeutic benefit to a subject. For example, a “therapeuticallyeffective amount of the active agent” to a subject is an amount of theactive agent that induces, ameliorates or causes an improvement in thepathological symptoms, disease progression, or physical conditionsassociated with the disease affecting the subject.

As used herein, the term “treating” a disorder or a condition refers toreversing, alleviating or inhibiting the process of one or more symptomsof such disorder or condition.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a subject according to theinvention is a human.

Pharmaceutical composition

Another object of the invention is a pharmaceutical composition for usein the treatment of cancer comprising:

-   -   a) at least one soluble peptide according to the invention; or    -   b) at least one acids nucleic according to the invention; or    -   c) at least one expression vector according to the invention or;    -   d) at least one host cell according to the invention;    -   e) and a pharmaceutically acceptable carrier.

In one embodiment, said pharmaceutical composition comprises at leastone soluble peptide having the sequence SEQ ID NO: 1.

In another embodiment, said pharmaceutical composition comprises afunction-conservative variant thereof of the peptide having the sequenceSEQ ID NO: 1.

In still another embodiment, said pharmaceutical composition comprisesthe peptide PKHB1.

Any therapeutic agent of the invention as above described may becombined with pharmaceutically acceptable excipients, and optionallysustained-release matrices, such as biodegradable polymers, to formtherapeutic compositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route ofadministration, the dosage and the regimen naturally depend upon thecondition to be treated, the severity of the illness, the age, weight,and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for atopical, oral, intranasal, intraocular, intravenous, intramuscular orsubcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The doses used for the administration can be adapted as a function ofvarious parameters, and in particular as a function of the mode ofadministration used, of the relevant pathology, or alternatively of thedesired duration of treatment.

To prepare pharmaceutical compositions, an effective amount of apolypeptide or a nucleic acid according to the invention may bedissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The peptides thereof or the nucleic acid according to the invention canbe formulated into a composition in a neutral or salt form.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The preparation of more, or highly concentrated solutions for directinjection is also contemplated, where the use of DMSO as solvent isenvisioned to result in extremely rapid penetration, delivering highconcentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution may be suitably buffered and the liquid diluent first renderedisotonic with sufficient saline or glucose. These particular aqueoussolutions are especially suitable for intravenous, intramuscular,subcutaneous and intraperitoneal administration. In this connection,sterile aqueous media which can be employed will be known to those ofskill in the art in light of the present disclosure. For example, onedosage could be dissolved in 1 ml of isotonic NaCl solution and eitheradded to 1000 ml of hypodermoclysis fluid or injected at the proposedsite of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; time release capsules; and any other form currentlyused.

In one embodiment, the pharmaceutical composition may comprise cellsstably expressing a peptide or variant thereof according to theinvention. For example, the pharmaceutical composition may compriseHEK293T cells stably expressing the peptide of the inventionpolypeptide, or HCT116 cells stably expressing the peptide of theinvention. The cells may be encapsulated in alginate gel beads, asdescribed in Desille et al., 2001, 2002 and Mahler et al., 2003. Thisvectorization approach enables a localized delivery of the polypeptideof the invention.

Compositions of the present invention may comprise a further therapeuticactive agent. The present invention also relates to a kit comprising apolypeptide or a nucleic acid according to the invention and a furthertherapeutic active agent.

In one embodiment said therapeutic active agent is an anticancer agent.For example, said anticancer agents include but are not limited tofludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, platinum complexes such as cisplatin,carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine,etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase,doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel andpaclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide,nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine,vinca alkaloids such as vinblastine, vincristine and vinorelbine,imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors,phosphatase inhibitors, ATPase inhibitors, tyrphostins, proteaseinhibitors, inhibitors herbimycm A, genistein, erbstatin, andlavendustin A. In one embodiment, additional anticancer agents may beselected from, but are not limited to, one or a combination of thefollowing class of agents: alkylating agents, plant alkaloids, DNAtopoisomerase inhibitors, anti-folates, pyrimidine analogs, purineanalogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonaltherapies, retinoids, photosensitizers or photodynamic therapies,angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors,acell cycle inhibitors, actinomycins, bleomycins, anthracyclines, MDRinhibitors and Ca2+ ATPase inhibitors.

Additional anticancer agents may be selected from, but are not limitedto, cytokines, chemokines, growth factors, growth inhibitory factors,hormones, soluble receptors, decoy receptors, monoclonal or polyclonalantibodies, mono-specific, bi-specific or multi-specific antibodies,monobodies, polybodies.

Additional anticancer agent may be selected from, but are not limitedto, growth or hematopoietic factors such as erythropoietin andthrombopoietin, and growth factor mimetics thereof.

In the present methods for treating cancer the further therapeuticactive agent can be an antiemetic agent. Suitable antiemetic agentsinclude, but are not limited to, metoclopromide, domperidone,prochlorperazine, promethazine, chlorpromazine, trimethobenzamide,ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine,alizapride, azasetron, benzquinamide, bietanautine, bromopride,buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol,dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl,pipamazine, scopolamine, sulpiride, tetrahydrocannabinols,thiethylperazine, thioproperazine and tropisetron. In a particularembodiment, the antiemetic agent is granisetron or ondansetron.

In another embodiment, the further therapeutic active agent can be anhematopoietic colony stimulating factor. Suitable hematopoietic colonystimulating factors include, but are not limited to, filgrastim,sargramostim, molgramostim and epoietin alpha.

In still another embodiment, the other therapeutic active agent can bean opioid or non-opioid analgesic agent. Suitable opioid analgesicagents include, but are not limited to, morphine, heroin, hydromorphone,hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine,etoipbine, buprenorphine, mepeddine, lopermide, anileddine,ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil,sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan,phenazodne, pemazocine, cyclazocine, methadone, isomethadone andpropoxyphene. Suitable non-opioid analgesic agents include, but are notlimited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal,etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin,ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen,piroxicam and sulindac.

In yet another embodiment, the further therapeutic active agent can bean anxiolytic agent. Suitable anxiolytic agents include, but are notlimited to, buspirone, and benzodiazepines such as diazepam, lorazepam,oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1. B cells isolated from 35 different CLL patients (includingpatients with intrinsic resistance to the current therapeutics,including unmutated IGHV, complex karyotype and dysfunctional TP53 orATM genes) were cultured 1 h in the presence of an immobilized CD47 mAb(clone B6H12), the soluble TSP-1-derived peptide 4N1K (300 μM), or thenegative soluble control peptide 4NGG (4N1K mutated in two amino acids;sequence: KRFYGGMWKK, SEQ ID NO:2). The percentage of cells that exposephosphatidylserine (measured with Annexin-V-FITC) was recorded andexpressed as a plot.

FIG. 2. (A) B cells isolated from a panel CLL patients or from controlvolunteers were pre-incubated (1 h) or not with 50 μM of the broadcaspase inhibitor z-VAD.fmk and cultured 1 h in the presence of 4N1K(300 μM) or the negative control peptide 4NGG (4N1K mutated in two aminoacids; sequence: KRFYGGMWKK, SEQ ID NO:2). The percentage of cells thatexpose phosphatidylserine (measured with Annexin-V-FITC) was recordedand expressed as a plot. ***, p<0.001 (different response to 4N1K in CLLand normal B cells). NS, Not significant difference in 4N1K-induced PCDbetween CLL cells treated or not with the caspase inhibitor ZVAD. (B) Apanel of B cells from CLL patients, analyzed by functional P53 activity,were cultured 1 h in the presence of 4N1K as above. PCD was measuredwith Annexin-V-FITC. NS, Not significant difference in PCD response to4N1K between CLL cells presenting functional and non-functional TP53.

FIG. 3. Electron micrographs of CLL cells untreated (control) orincubated with 4N1K (CD47). Upper panels demonstrate a typical exampleof the mitochondrial (MT) and ER normal morphology. Lower panels showthe mitochondrial morphology and ER dilation observed in CD47mAb-treated cells.

FIG. 4. Free Ca2+ mobilization assessed with Fura-2 AM by Till photonicsin B-lymphocytes from control volunteers (n=4; on the left) and B cellsfrom representative CLL patients (n=4; on the right) after CD47stimulation with 4N1K. Ionomycin (Iono), which was used as a positivecontrol, signals the maximum of Ca2+ monitored. Importantly, in CLL theCa2+ mobilization induced by 4N1K does not come back to basal level (asobserved in normal B cells) and underlined a calcium overload in4N1K-treated CLL cells.

FIG. 5. The same panel of B cells from CLL patients depicted in FIG. 2was pre-incubated or not with the calcium chelator BAPTA-AM (20 μM)prior to induction of PCD by 4N1K as above. ***, p<0.001. Note that thetreatment with BAPTA abrogates 4N1K-mediated PCD.

FIG. 6. (A) B cells isolated from CLL patients were left untreated(control) or cultured 1 h in the presence of 4N1K or the 4N1K-derivativepeptide PKHB-1 at the indicated concentration. The percentage of cellsthat expose phosphatidylserine (measured with Annexin-V-FITC) wasrecorded and expressed as a plot. Data are the means of eightindependent experiments. (B) In a similar experiment B-lymphocytes from14 different CLL patients, including individuals with unmutated IGHV,complex karyotype and dysfunctional TP53, were left untreated (control)or cultured 1 h in the presence of 4N1K (300 μM) or the 4N1K-derivativepeptide PKHB-1 (150 μM). PCD was measured by Annexin-V-FITC labelling ina flow cytometer. (C) Cell death was determined as described in A in4N1K or PKHB1-treated CD19⁺/CD5⁻ (residual B-cells) and CD19⁺/CD5⁻B-lymphocytes (CLL cells) identified by flow cytometry from each CLLpatient. Data are presented as the mean±s.d. (n=8 patients). (D) Celldeath was measured by Annexin V-positive staining in 4N1K orPKHB1-treated CLL cells pre-incubated or not with the intracellularcalcium chelator BAPTA-AM. Data are presented as the mean±S.D. (n=10patients).

FIG. 7: (A and B) NSG mice (n=8 per group) transplanted subcutaneouslywith MEC-1 cells received three times a week intraperitoneal injectionof 4N1K, PKHB1 or PBS (Control). Tumor volume was measured with acalliper and plotted as a graph. (C) Tumor growth after treatments wasalso visualized in a photograph by glucose uptake 2-DG. Colour scaleindicates fluorescence intensity in arbitrary units.

FIG. 8: 4N1K and PKHB1 peptides stability were evaluated in human serumat 37° C. for 6 h. The relative concentrations of the remaining solublepeptides were analyzed by HPLC, by the integration of the absorbance at220 nm as a function of retention time.

TABLE 1 PCD induction in cancer cell lines Cell death induction CancerCell Line via CD47 Acute T cell leukemia JURKAT + Acute lymphoblasticleukemia CEM + Adenocarcinoma MDA-MB-231 + Breast adenocarcinoma MCF-7 +Breast adenocarcinoma AU-565 + Breast epithelial cells with HBL-100 +transformed morphology Burkitt's lymphoma RAJI + Burkitt's lymphomaRAMOS + B lymphocytic cell line RPMI 8226 + B lymphocytic cell line RPMI8866 + Cervical cancer HELA + Chronic Lymphocytic Leukemia MEC-1 +Diffuse histiocytic lymphoma U937 + Immunoblastic B cell JM-1 + lymphomaOvarian Carcinoma OV10 + Lung carcinoma A549 + Prostrate cancerLNCAP + + = more than 35% of cell death induction, assessed by Annexin Vlabeling, after 1 h of stimulation with 400 μM of 4N1K (means of sixindependent experiments). No cell death was recorded with the controlpeptide 4NGG.

TABLE 2 PCD induction in primary cancer cells Cell death induction viaCancer CD47 Ovarian carcinoma + Breast carcinoma + Colon carcinoma +Bladder carcinoma + Glioblastoma + Hepatocellular carcinoma + Prostatetumor cells + Glioma + Follicular lymphoma + Mantle cell lymphoma +Diffuse large B cell lymphoma + Chronic Lymphocytic Leukemia + MarginalB cell lymphoma + Lymphoplasmacytic lymphoma + Hairy cell leucemia(HCL) + Acute T cell leukemia + + = more than 35% of programmed celldeath induction via CD47 receptor.

EXAMPLES Example 1 Use of 4N1K as Therapeutic Compound

Material & Methods

Patients, B-Cell Purification, and Culture Conditions.

After authorized consent forms fresh blood samples will be collectedfrom CLL patients diagnosed according to classical morphological andimmunophenotypic criteria at Pitié-Salpêtrière Hospital (Serviced'Hématologie Biologique). Normal B lymphocytes will be obtained fromEFS (Etablisement Français du Sang). The Institutional Ethics Committeeat Pitie-Salpetriere Hospital approved the present study. Mononuclearcells were purified from blood samples using a standard Ficoll-Hypaquegradient, and B cells were positively or negatively selected by magneticbeads coupled to anti-CD19 monoclonal antibody (positive selection) orto anti-CD16, CD3, and CD14 monoclonal antibodies (negative depletion)(Miltenyi Biotech). No changes were found in the cell death response ofpositively or negatively selected cells. Purified B-lymphocytes werecultured in complete medium (RPMI 1640 medium supplemented with 10%fetal calf serum, 2 mM L-glutamine, and 100 U/mlpenicillin-streptomycin). Unless specified, reagents were fromSigma-Aldrich.

Cell Death Induction and Inhibition.

To induce cell death B-lymphocytes were cultured 1 h with the TSP-1derived peptide 4N1K (300 μM; sequence KRFYVVMWKK, SEQ ID NO:1), thenegative control peptide 4NGG (300 μM; sequence KRFYGGMWKK, SEQ IDNO:2), the newly developed peptide PKHB1 (150 μM; sequence(D)K-R-F-Y-G-G-M-W-(D)K) (formula I) or an anti-CD47 mAb (in solubleconditions or in immobilized precoated plates; 5 μg/ml; clone B6H12).Alternatively, B-cells were pre-treated for 30 min before the inductionof cell death with the calcium chelator BAPTA-AM (20 μM), thecaspase-inhibitor Z-VAD.fmk (50 μM).

Flow Cytometry.

We used Annexin V-FITC (BD Biosciences) for the assessment ofphosphatidylserine (PS) exposure. Data analysis was carried out in aFACSCanto II (BD Biosciences) on the total cell population (10,000cells). Data were analyzed using FlowJo software (TreeStar).

Ca2+ Measurements.

Cells, adhered in Polylysine coated glass-bottom Petri dishes, wereloaded with Fura-2 AM (1 μM/30 min/37° C.). Cells were then excited bywavelengths of 340 and 380 nm and fluorescence emission of several cellswas simultaneously recorded at 510 nm at a frequency of 1 Hz using adual excitation fluorometric imaging system (TILL-Photonics) controlledby TILL-Vision software. Signals were computed into relative ratio unitsof the fluorescence intensity of the different wavelengths (340/380 nm).Individual fluorescence values were then analyzed with Origin softwareto normalize the fluorescence with the first value according to theequation (F/F0)−1, where ‘F’ is the fluorescence at specific time pointand ‘F0’ is the fluorescence at time 0.

Electron Microscopy.

Cells were fixed with 2% glutaraldehyde in phosphate buffer (pH 7.4) for2 h at RT, washed, and postfixed in 2% OsO4 before being embedded inDurcupan™. Analysis was performed with a transmission electronmicroscope (Carl Zeiss Microlmaging), on ultrathin sections stained withuranyl acetate and lead citrate.

In Vivo 4N1K and PKHB-1 Treatments in a Localized CLL Xenograft Model.

3×10⁶ MEC-1 cells were injected subcutaneously into NOD scid gamma (NSG)mice (Charles River). When the tumor volume reached 0.1 cm³, mice wereinjected intraperitoneally three times per week with 4NIK or PKHB1 (400μg in 200 μl PBS). Tumor size was measured every 2-3 days with acalliper and tumor volume, calculated using the formula (length xwidth)/2, was expressed as mm³. Alternatively, at the end of treatmentmice were injected into the sinus retro-orbital with XenoLight RediJect2-DG-750 Probe (Caliper) to visualize tumor cell glucose uptake, whichreflects cell proliferation. Fluorescence was measured by In vivoimaging system FX Pro (Kodak) and pictures were analysed with theCarestream MI software.

Statistical Analysis.

The significance of differences between experimental data was determinedusing Student's t test for unpaired observations or for comparativeanalysis of different groups Mann-Whitney test as described, usinggraphpad prism software.

Results

Effect of 4N1K Peptide on B Cell from CLL Patients:

The inventors have recently identified that the use of the 4N1K peptide(SEQ ID NO:1) which mimics the C-terminal domain of TSP-1, does notneeds to be immobilized to induced caspase-independent PCD as comparedto the anti-CD47 mAb (FIG. 1), and that even in CLL cells fromrefractory patients (ATM or TP53 mutated/deleted, which are refractoryto the most common drug treatments used against this leukemia, such asanti-CD20, fludarabine or cladribine; FIG. 2).

The previous results of the inventors indicate that CD47-mediatedcaspase-independent PCD proceeds via the induction of atypicallyregulated mitochondrial alterations that are independent from thecaspase-dependent apoptotic regulators usually blocked in CLL. Indeed,these results on CD47 are the first published incidence of massivemitochondrial alterations induced without the involvement of caspases(FIG. 3). In addition, experiments performed in B cells from controldonors and from a large number of CLL patients (more than 150 to date),have shown that CD47 ligation induces cell death rapidly and with ahigher efficacy in CLL cells than in normal B cells. And that, even inB-leukemic cells from drug refractory CLL patients.

Caspase-Independent PCD Induced by 4N1K in CLL

1. Intracellular organelles are key elements in the regulation ofprogrammed cell death, the inventors have performed an ultrastructuralstudy of CLL cells after CD47 ligation. After CD47 triggering, theendoplasmic reticulum (ER) dilates and mitochondria swells and undergoesa morphological change (FIG. 3). These alterations, typical hallmarks ofthe Ca2+-mediated PCD, seem to indicate that mitochondria and ER areinterconnected both physically and physiologically, mainly through thecalcium ion: (i) Mitochondria, the main source of cellular adenosinetriphosphate, also modulate and synchronize ER Ca2+ signaling; (ii)Stimuli that generate inositol 1,4,5-trisphosphate (IP3) cause releaseof Ca2+ from the ER, which is rapidly taken up by closely juxtaposedmitochondria.

Using Till photonics Imaging Technology, the inventors have confirmedthat the ligation of CD47 in CLL cells is followed by an intracellularfree Ca2+ mobilization. Strikingly, compared to normal B cells, therecorded intracellular Ca2+ mobilization is stronger and sustained inleukemic B cells but not in normal B-lymphocytes (FIG. 4). Indeed, inCLL, Ca2+ mobilization induced by 4N1K does not come back to the basallevel indicating a Ca2+ overload particularly in these cells. Theseresults correlate to the different sensibility of CLL and normal B cellsto CD47-mediated PCD (FIG. 2).

2. Pre-treatment of B cells with the intracellular calcium chelatorBAPTA-AM blocks CD47-mediated PCD (FIG. 5). This indicates that the Ca2+released into the cytosol after 4N1K-triggering plays a pivotal role inthis type of caspase-independent PCD in CLL cells.

Importantly, when the inventors analyze PCD and intracellular calciummobilization in CLL cells from patients with a good prognosis andpatients with refractory CLL (unmutated IGHV gene status, high levels ofthymidine kinase, soluble CD23, CD38, and ZAP-70 expression, and ATM orTP53 mutation/deletion), they find that the cells from patients with abad prognosis, which are resistant to caspase-dependent PCD induced byanti-CD20, fludarabine or cladribine, present similar levels ofCD47-mediated PCD and, consequently, similar levels of intracellularCa2+ mobilization after CD47-triggering. These results indicate that thecalcium pathway enabled by CD47 is functional even in CLL cells fromrefractory patients (with a defective caspase-dependent PCD pathway).Thus, 4N1K-mediated caspase-independent PCD occurs more efficiently inCLL cells than in B-lymphocytes from healthy donors and represents abetter means of inducing death than caspase-dependent PCD.

3. The inventors have shown that the ligation of the CD47 receptorinduces PCD more efficiently in CLL cells than in B-lymphocytes fromhealthy donors and represents a better means of inducing death thancaspase-dependent PCD. Thus, the inventors have hypothesized that theCa2+-mediated caspase-independent PCD pathway induced by CD47 could beused to eliminate in vivo B leukemic cells. To substantiate thishypothesis, the inventors have developed a xenograft mouse model andconducted in vivo experiments (Materials and Methods for details andFIG. 7). Contrary to controls, the IP injection of the CD47-ligand 4N1Kderivatives induce a low reduction in tumor volume.

These new findings are particularly interesting given the potentialapplication of a 4N1K derivatives such as PKHB1, therapy as a treatmentfor CLL. In this way, it is important to note that: (i) the 4N1Kderivatives peptides do not exert toxicity in mice, and (ii) 4N1Kderivatives-treatment provokes the external exposure of “eat mesignals”, such as phosphatidylserine or calreticulin (data not shown).This facilitates the subsequent elimination of dying cells byprofessional phagocytes.

Effect of 4N1K Peptide on Cancer Cell Lines and Primary Cancer Cells:

The inventors show that stimulation with 4N1K induces more than 35% ofcell death induction on cancer cell lines and primary cancer cellswhereas 4NGG, the control peptide, is inefficient in inducingcaspase-independent PCD (Tables 1 and 2).

These results show that the soluble peptide 4N1K derivatives could beused in a large variety of tumor models.

Example 2 Synthesis of New Soluble Peptides

The structure of 4N1K peptide is as followed:

4N1K is an undecapeptide with the following sequence:K-R-F-Y-V-V-M-W-K-K (SEQ ID NO:1).

To improve peptide solubility, stability and pharmacological properties,the 4N1K is modified by chemical modifications that are established onthe following 2 models:

Model 1:

-   -   A1-A2-A3-A4-A5-A6-A7-A8-A9-A10

Model 2:

In model peptide 1, A (1 to 10) correspond to nothing and/or naturalamino acids and/or non natural amino acids or amino acids derivatives asfor example prolinoamino acids [Mothes C et al., 2008], beta-amino acids[Moumne R et al., 2007], cyclopropylamino acids [Joosten A et al.,2009], N-methylamino acids [Sagan S et al., 2004], and/or alpha-alphadisubstituted amino acids, and/or disubstituted beta-amino acids(Beta2,2, beta3,3 ou beta2,3) and or aza-amino acids (Proulx, C et al.,2011) or azidolysine [Larregola M et al., 2001]. The absoluteconfigurations of the stereogenic centers are not indicated since allthe amino acids are natural amino acids or synthetically obtained aminoacids, enantiomerically pur from (L) or (D) series or used as racemate.

For model 2, the absolute configurations of the stereogenic centers arenot indicated since all the amino acids are natural amino acids orsynthetically obtained amino acids, enantiomerically pur from (L) or (D)series or used as racemate.

The R groups (1 to 10) correspond to amino acids side chains that can benatural or synthetic amino acids side chains. For the glycine residue,this side chain correspond to a hydrogen atom.

P1 and P2 correspond to functions respectively on the N-terminal andC-terminal sides of the peptides.

Thus, P1 can be an amine function (P1=—NR11, where R11 is a hydrogenatom or an alkyle chain (similar to methyl, ethyl or benzyl), acarboxylic function (P1=—CO₂H), a carboxamide function (P1=—CONR12R13,where R12 and R13 are hydrogen atoms and/or alkyle chains (similar tomethyl, ethyl or benzyl) or an amine function, or an azido function(N3).

Thus, P2 can be an amine function (P2=—NR11, where R11 is a hydrogenatom or an alkyle chain (similar to methyl, ethyl or benzyl), acarboxylic function (P2=—CO₂H), a carboxamide function (P2=—CONR12R13,where R12 and R13 are hydrogen atoms and/or alkyle chains (similar tomethyl, ethyl or benzyl) or an amine function, or an azido function(N3).

Y groups (1 to 10) correspond to hydrogen atoms and/or methyl groups,and/or natural amino acids side chains.

Y1 can also be a protecting group such as an acetamide, a benzamide, abenzyloxycarbonyl, a tertbutyloxycarbonyl, aphenylfluorenylmethoxycarbonyl protecting groups.

Peptides and analogues are synthesized by SPPS or LPPS with Boc, Fmoc orZ strategies. The protonation states of all functions (amino groups,carboxylic functions, guanidinium . . . ) is depending on the synthesesand purifications procedures and can be different from the one indicatedon the schemes.

I—Short Analogues Based on Model 1: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10

To improve the solubility and the stability of the octapeptideR-F-Y-V-V-M-W-K (SEQ ID NO:3), the following chemical modifications arerealized:

a) Mixed Salts:

N-methylmorpholine and chlorhydrate or trifluoroacetylcarboxylate mixedsalts:

R7 refers to methionine, methionine sulfoxyde, methionine sulfone oralanine or butylglycine or lysine side chain.

A2 refers to lysine or azidolysine or arginine or bis-ornithine[Aussedat B et al., 2006] or bis-arginine or beta-2-homolysine, orbeta-2-homoarginine or beta-2-bis-homoornithine orbeta-2-bis-homoarginine. The N-terminal amine function can be free orprotected by Boc, Fmoc or Cbz groups, and/or can be N-methylated.

A9 refers to a lysine or arginine or bis-ornithine or bis-arginine or abeta-3-homolysine, or a beta-3-homoarginine or abeta-3-bis-homoornithine or a beta-3-bis-homoarginine. The A9 carboxylicfunction is free or protected as a carboxamide (P2=—CONR12R13, where R12and R13 are hydrogen atoms and/or alkyls groups such as methyl, ethyle,benzyl groups and derivatives).

Free or N-methylated amine functions can be obtained as Chlorhydrate orTFA salts (n is variable, depending on the nature of A2, R7 or A9).

Free carboxylic function can be obtained (but not necessarily) asN-methylmorpholine salts (p=1).

PKHB3 is an example of peptide from these series where A2 and A9correspond respectively to beta-2-homoarginine and beta-3-homolysine:

b) Tetraphenylborate Salts:

R7 corresponds to methionine, methionine sulfoxyde, methionine sulfoneor alanine or butylglycine or lysine side chains.

A2 corresponds to lysine or azidolysine or arginine or bis-ornithine orbis-arginine or a beta-2-homolysine, or a beta-2-homoarginine or abeta-2-bis-homoornithine or a beta-2-bis-homoarginine. The freeN-terminal amine function can also be protected by a Boc, Fmoc or Cbzgroups, and/or N-methylated.

A9 corresponds to lysine or arginine or bis-ornithine or bis-arginine orbeta-3-homolysine, or beta-3-homoarginine or beta-3-bis-homoornithine orbeta-3-bis-homoarginine. The carboxylic function of A9 is free orprotected as a carboxamide (P1=—CONR12R13, where R12 and R13 arehydrogen atoms and/or alkyl groups such as methyl, ethyl, benzylderivatives).

The free or N-methylated amine functions are obtained astetraphenylborate salts, m being variable, depending on A2, R7 or A9.

The structure and the syntheses of the peptides below are given asexamples:

c) N and C-Termini Protections:

The peptides described above are also prepared with N- and C-terminiprotecting groups.

d) Various Chemical Modifications:

-   -   A2-A3-A4-A5-A6-A7-A8-A9

N-methyl amino acids are introduced on various positions as describedhere below:

-   -   R7 is described above.

e) (D)-Amino Acids:

The introduction of (D)-amino acids in peptide sequences stabilizepeptides towards proteolytic degradations and thus enhance itspharmacological properties. (D)-amino acids are introduced on the Nterminus and/or on the -terminus and/or instead of one, two, three,four, five, six or seven residues in peptide sequence:

(

)R-F-Y-V-V-M-W-K R-(

)F-Y-V-V-M-W-K R-F-(

)Y-V-V-M-W-K R-F-Y-(

)V-V-M-W-K R-F-Y-V-(

)V-M-W-K R-F-Y-V-V-(

)M-W-K R-F-Y-V-V-M-(

)W-K R-F-Y-V-V-M-W-(

)K

f) Retro-Inverso Sequences:

The retro-inverso sequences led to peptides partially or fully modifiedwith (D)-amino acids keeping however the spatial orientations of crucialamino acids side chains. The following peptides are prepared,incorporating some chemical modifications in order to maintain peptidepolarity, around diamine and diacid, introduced respectively on theC-terminus and N-terminus sides:

R4 correspond to methionine or alanine or butylglycine or lysine sidechains.

g) Some Analogues Based on Model 1: A1-A2-A3-A4-A5-A6-A7-A8-A9-A10Incorporating Modified Amino Acids:

A1 and A10 can correspond to (D)-lysine residues or (D)-arginineresidues or beta-2 and/or beta-3 homolysine and/or beta-2 and/or beta-3homoarginine or beta-2,2 or beta-3,3-homolysine or beta-2,2 orbeta-3,3homoarginine PKHB4 (formula III) is an example from this serieswhere A10 have been replaced by a (D)-Lysine residue:

PKHB11 is another example of peptide from this series where both A1 andA2 residues have been replaced by the beta-2,2 homolysine and a D-lysinehas been introduced in C-terminal position of the peptide (see forexample PKHB11 (formula (IV)):

A2 can correspond to arginine or lysine residues.N-methylated amino acids or cyclopropylamino acids can be introduced inthe sequence in position A2 and/or A3 and/or A4 and/or A5 and/or A6and/or A7 and/or A8 and/or A9.A prolinovaline can be introduced in position A5 and/or A6. PKHB9(formula (V)) and PKHB10 (formula (VI)) are both example of peptide fromthis series:

A prolinomethionine can be introduced in position A7.A prolinotryptophane or prolinohomotryptophane can be introduced inposition A8.A prolinolysine can be introduced in position A9.

II—Analogues Based on Model 2:

a) Protections of N- and C-Termini of the Peptide

P1 is an amine function (P1=—NR11, where R11 is a hydrogen atom or analkyl chain such as a methyl group for example) or a carboxylic acid(P1=—CO₂H) or an azido group (N3).

P2 can be an amine function (P2=—NR11, where R11 is a hydrogen atom oran alkyl chain such as a methyl group for example) or a carboxylic acid(P1=—CO₂H) or an azido group (N₃).

The following peptide is given as an example:

An azido group is present on the N-terminal side. An azido-lysine with Sor R configuration is introduced. The R2 group corresponds to lysine orarginine side chains. R7 group corresponds to methionine side chain,methionine sulfoxyde side chain, methionine sulfone side chain or amethyl, or an n-butyl or lysine side chain.

b) Amino Acids Sequence Modifications

(D) Residues:

The introduction of (D)-amino acids in peptide sequences stabilizepeptides towards proteolytic degradations and thus enhance itspharmacological properties. (D)-amino acids are introduced on the Nterminus and/or on the C-terminus and/or instead of one, two, three,four, five, six, seven, eight or nine residues in peptide sequence:

The retro-inverso sequences lead to peptides partially or fully modifiedwith (D)-amino acids keeping however the spatial orientations of crucialamino acids side chains. The following peptides are prepared,incorporating some chemical modifications in order to maintain peptidepolarity, around diamine and diacid, introduced respectively on theC-terminus and N-terminus sides:

Inverso Peptide: (

)K-(

)R-(

)F-(

)Y-(

)V-(

)V-(

)M- (

)W-(

)K-(

)K Retro-inverso peptide: (SEQ ID NO: 5) (

)K-(

)K-(

)W-(

)M-(

)V-(

)V-(

)Y- (

)F-(

)R-(

)K

Specific chemical modifications are introduced in retro-inverso peptidesequence in order to keep peptide polarity. The following peptides areprepared:

In this peptide, (D)-amino acids are introduced instead of (L)-aminoacids, excepted on the N-terminus and C-terminus where diacide(N-terminus) and diamine (C-terminus) are introduced keeping 4N1Kpeptide polarity.

III—Polymer Analogues

Polymers (dimers or trimers) analogues are obtained by reacting theazidopeptides (short or long analogues) through Huisgen typecycloaddition reactions:

For example, trimer analogues are obtained by reacting the azidopeptides(short or long analogues) with tripropargylamine as followed:

The R group corresponds to sequences of azidopeptides described hereabove.

V—Test of Generated Soluble Peptides

1.—To verify whether the newly generated soluble peptides induce thesame type of caspase-independent PCD than the initially characterizedpeptide 4N1K, a flow cytometry analysis (such as described in FIG. 2) isperformed with each peptide. A comparison of the results obtained intreatments with/without the caspase inhibitor Z-VAD.fmk corroborate thatthe mode of cell death induced by the 4N1K-derived peptides iscaspase-independent. Additionally, a time-course and a dose-responseperformed in different tumor cells determine the optimal conditions foreach peptide and each malignant cell type.

2.—As shown in FIGS. 3 and 4 the induction of 4N1K induces a potent andsustained increase of calcium mobilization. This Ca²⁺ overloaddramatically changes the morphological structure of organelles such asmitochondria driving cells in apoptosis. Therefore, using our ionmeasurement platform, we assess Ca²⁺-mobilization in fura-2 loaded CLLcells triggered by the 4N1K-derived peptides.

3.—Since we have shown that the injection of the 4N1K derivatives suchas PKHB1 peptide may be used to eliminate B leukemia cells in vivo (FIG.7), we use the same approach and test the newly synthesized and invitro-validated peptides. The objective is to corroborate whether thepeptides generated are efficient in inducing tumor regression in vivo.To do so, the CLL established MEC1 B-cells are injected subcutaneouslyinto the flanks of NSG (NOD scid gamma) mice, one of the most suitableimmunodeficient strain. The mice with established lymphoma are treated 3times a week for 4 weeks by intraperitoneal injections of the 4N1Kderivatives or PBS control, and the development of the tumor is followedby caliper measurement and by a non-invasive optical analysis performedin a Kodak in vivo-imaging system FX-Pro using the 2-DG-750 probe (Tumorvolume is monitored every other day).

Example 3 Use of PKHB1 as Therapeutic Compound

The PKHB1 compound correspond to the peptide 4N1K (SEQ ID NO:1) withamino acids C-terminal and N-Terminal (A1 and A10 of SEQ ID NO:1) in(D)-amino acids (dextrorotary amino acids) version as explained inexample 2 part II b)).

The PKHB1 peptide is not degradated in human serum experiments and hasproven to be stable over 6 hours (FIG. 8).

The PKHB1 peptide shows clearly a better efficiency than the 4N1Kpeptide on B cells isolated from CLL patients (FIG. 6A). This wasverified in a cohort of 14 CLL patients with diverse prognosticfeatures, including unmutated IGHV, complex karyotype and dysfunctionalTP53 (FIG. 6B, 4N1K induced about 45% of cell death in CLL cells at 300μM, whereas PKHB1 induced a similar rate of death at 150 μM). Moreover,as 4N1K, PKHB1 induced cytotoxicity in malignant cells but not in normalB-lymphocytes (FIG. 6C, PKHB1 significantly killed the CD19⁺/CD5⁺ tumorcells, but the residual CD19⁺/CD5⁻ B-lymphocytes were resistant to thispeptide, n=8). Finally, we have corroborated that pre-treatment of CLLcells with BAPTA-AM, an intracellular Ca²⁺ chelator, significantlydecreased PKHB1-mediated PCD in CLL cells (FIG. 6D, n=10). These resultsconfirmed that PKHB1 induced cell death in CLL in a similar way than4N1K, via an intracellular calcium mobilization. Overall, our results onPKHB1 confirmed that the introduction of (D)-amino acids in the peptidesequence of 4N1K enhanced its pharmacological cell death activity.

Since the PKHB1 had a better stability and in vitro effect, we testedthe in vivo role of PKHB1 in tumor growth. As depicted in FIG. 7, onlyfew days after PKHB1 injection, we observed a reduction of both thetumor growth and glucose uptake compared to control mice. These in vivoresults emphasize the use of the 4N1K derivatives, like PKBH1, astherapeutic tools for the eradication of tumor cells.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. A soluble peptide of formula (II):